Protein-Stabilized Palm-Oil-in-Water Emulsification Using Microchannel Array Devices under Controlled Temperature.

Microchannel (MC) emulsification for the preparation of monodisperse oil-in-water (O/W) and water-in-oil-in-water (W/O/W) emulsions containing palm oil as the oil phase was investigated for application as basic material solid/semi-solid lipid microspheres for delivery carriers of nutrients and drugs. Emulsification was characterized by direct observation of droplet generation under various operation conditions, as such, the effects of type and concentration of emulsifiers, emulsification temperature, MC structure, and flow rate of to-be-dispersed phase on droplet generation via MC were investigated. Sodium caseinate (SC) was confirmed as the most suitable emulsifier among the examined emulsifiers, and monodisperse O/W and W/O/W emulsions stabilized by it were successfully obtained with 20 to 40 µm mean diameter (dm) using different types of MCs.

Follistatin expression in the central nervous system of the adult rat.

Follistatin was initially cloned as a monomeric polypeptide that inhibits the release of follicle-stimulating hormone. Although follistatin also plays pivotal roles in skeletal muscle hypertrophy and immunoregulation in the epididymis, little information is available regarding follistatin function in the adult central nervous system (CNS). Hence, we investigated follistatin expression in the adult rat CNS using immunohistochemistry. Follistatin was intensely expressed in most neurons and their axons. Furthermore, oligodendrocytes, ependymal cells, and some astrocytes also expressed follistatin protein. These data indicate that follistatin is widely expressed throughout the adult CNS. The abundant expression of follistatin in the adult brain suggests that this protein plays important roles in the CNS.

Acoustic Histology with Specific Dyes and Antibodies.

The present study aims to identify specific staining methods for acoustic histology. We compared attenuation-of-sound (AOS) images from scanning acoustic microscopy (SAM) with light microscopy (LM) images. Ethanol-fixed tissue or cytology samples and formalin-fixed surgical or autopsy specimens were examined. Nuclei, collagen, elastic fibers and polysaccharides and various antigens, including cell surface, cytoplasmic, nuclear and stromal substances, were observed. Samples with various fixation methods were used. Hematoxylin staining had significantly higher AOS values in accordance with staining duration. Specific staining for collagen, elastic fibers and polysaccharides increased the AOS values of the specific substance. Using diaminobenzidine tetrahydrochloride in NiCl2 solution as a substrate for horseradish peroxidase increased the AOS values to those suitable for acoustic immunostaining. Collagenase digestion after collagen staining decreased AOS values, reflecting collagen density and distribution. Staining with specific dyes or acoustic immunostaining enabled the histologic localization of specific substances by SAM, similar to LM.

Mechanical Weakness of Thoracic Aorta Related to Aging or Dissection Predicted by Speed of Sound with Collagenase.

Scanning acoustic microscopy reveals information on histology and speed of sound (SOS) through tissues. Slower SOS corresponds to lower stiffness. The aim of the present study was to investigate whether SOS values reflect the degree of degeneration with aging or dissection and whether enzymatic digestion susceptibility is distinct. The SOSs of media other than the atheromatous areas of normal and surgical dissections were measured and compared using medial degeneration grade (MDG) scores. To evaluate the damage rate, SOS was assessed after collagenase digestion. SOS scores negatively correlated with aging and MDG scores. Dissected aortas had higher SOS and MDG scores without age correlation. Collagenase digestion was present in all aortas, but older aortas were more injured than younger aortas. Dissected aortas were more vulnerable to collagenase. Older and dissected aortas expressed specific extracellular matrix components to compensate for mechanical weakness. The present method can evaluate mechanical weakness corresponding to histology to investigate the cause of rupture.

Pinacidil, a KATP channel opener, stimulates cardiac Na+/Ca2+ exchanger function through the NO/cGMP/PKG signaling pathway in guinea pig cardiac ventricular myocytes.

Pinacidil, a nonselective ATP-sensitive K+ (KATP) channel opener, has cardioprotective effects for hypertension, ischemia/reperfusion injury, and arrhythmia. This agent abolishes early afterdepolarizations, delayed afterdepolarizations (DADs), and abnormal automaticity in canine cardiac ventricular myocytes. DADs are well known to be caused by the Na+/Ca2+ exchange current (INCX). In this study, we used the whole-cell patch-clamp technique and Fura-2/AM (Ca2+-indicator) method to investigate the effect of pinacidil on INCX in isolated guinea pig cardiac ventricular myocytes. In the patch-clamp study, pinacidil enhanced INCX in a concentration-dependent manner. The half-maximal effective concentration values were 23.5 and 23.0 µM for the Ca2+ entry (outward) and Ca2+ exit (inward) components of INCX, respectively. The pinacidil-induced INCX increase was blocked by L-NAME, a nitric oxide (NO) synthase inhibitor, by ODQ, a soluble guanylate cyclase inhibitor, and by KT5823, a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor, but not by N-2-mercaptopropyonyl glycine (MPG), a reactive oxygen species (ROS) scavenger. Glibenclamide, a nonselective KATP channel inhibitor, blocked the pinacidil-induced INCX increase, while 5-HD, a selective mitochondria KATP channel inhibitor, did not. In the Fura-2/AM study pinacidil also enhanced intracellular Ca2+ concentration, which was inhibited by L-NAME, ODQ, KT5823, and glibenclamide, but not by MPG and 5-HD. Sildenafil, a phosphodiesterase 5 inhibitor, increased further the pinacidil-induced INCX increase. Sodium nitroprusside, a NO donor, also increased INCX. In conclusion, pinacidil may stimulate cardiac Na+/Ca2+ exchanger (NCX1) by opening plasma membrane KATP channels and activating the NO/cGMP/PKG signaling pathway.

Evaluation of aging, diabetes mellitus, and skin wounds by scanning acoustic microscopy with protease digestion.

Scanning acoustic microscopy (SAM) can assess tissue stiffness by calculating the speed of sound (SOS) through tissues. SOS increases as tissue stiffness increases. Sensitivity to protease digestion depends on protein type, concentration, and modification. We analyzed the SOS images of formalin-fixed paraffin-embedded skin sections from elderly, young, diabetic, and nondiabetic subjects, as well as chronic and acute wounds. SAM provided high-resolution histology similar to LM and revealed characteristic SOS alteration following pepsin treatment. SOS values of dermis samples from elderly subjects (especially females) were lower than those of younger adults, which was indicative of age-related dermal softening and loosening. SOS values of elderly females were lower than those of younger females and elderly males. Dermal SOS showed a positive correlation with epidermal thickness. SOS values of epidermis of elderly subjects were higher than those of younger adults and showed a rapid decline 0.5h after protease digestion. Reticular dermis of diabetic patients exhibited greater pepsin resistance than that of nondiabetic patients. Chronic wounds exhibited greater SOS values and pepsin resistance than acute wounds. SOS variation with aging, diabetes mellitus, and wound fibrosis reflected histological and mechanical changes associated with senescence and disease duration. Epidermal thickness reflects age-related changes in dermal stiffness.

Suppressive Effect of Carvedilol on Na+/Ca2+ Exchange Current in Isolated Guinea-Pig Cardiac Ventricular Myocytes.

BACKGROUND AND AIMS: Carvedilol ((+/-)-1-(carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol), a ß-adrenoceptor-blocker, has multi-channel blocking and vasodilator properties. This agent dose-dependently improves left ventricular function and reduces mortality in patients with arrhythmia and chronic heart failure. However, the effect of carvedilol on the cardiac Na+/Ca2+ exchanger (NCX1) has not been investigated. METHODS AND RESULTS: We examined the effects of carvedilol and metoprolol, 2 ß-blockers, on Na+/Ca2+ exchange current (INCX) in guinea-pig cardiac ventricular cells and fibroblasts expressing dog cardiac NCX1. Carvedilol suppressed INCX in a concentration-dependent manner but metoprolol did not. IC50 values for the Ca2+ influx (outward) and efflux (inward) components of INCX were 69.7 and 61.5 µmol/l, respectively. Carvedilol at 100 µmol/l inhibited INCX in CCL39 cells expressing wild type NCX1 similar to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 µmol/l abolished ouabain-induced delayed afterdepolarizations. CONCLUSION: Carvedilol inhibited cardiac NCX in a concentration-dependent manner in isolated cardiac ventricles, but metoprolol did not. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.

Inhibitory effect of YM-244769, a novel Na+/Ca2+ exchanger inhibitor on Na+/Ca2+ exchange current in guinea pig cardiac ventricular myocytes.

Recently, YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) has been reported as a new potent and selective Na+/Ca2+ exchange (NCX) inhibitor by using various cells transfected with NCX using the 45Ca2+ fluorescent technique. However, the electrophysiological study of YM-244769 on NCX had not been performed in the mammalian heart. We examined the effects of YM-244769 on NCX current (INCX) in single cardiac ventricular myocytes of guinea pigs by using the whole-cell voltage clamp technique. YM-244769 suppressed the bidirectional INCX in a concentration-dependent manner. The IC50 values of YM-244769 for the bidirectional outward and inward INCX were both about 0.1 µM. YM-244769 suppressed the unidirectional outward INCX (Ca2+ entry mode) with an IC50 value of 0.05 µM. The effect on the unidirectional inward INCX (Ca2+ exit mode) was less potent, with 10 µM of YM-244769 resulting in the inhibition of only about 50 %. At 5 mM intracellular Na+ concentration, YM-244769 suppressed INCX more potently than it did at 0 mM [Na+]i. Intracellular application of trypsin via the pipette solution did not change the blocking effect of YM-244769. In conclusion, YM-244769 inhibits the Ca2+ entry mode of NCX more potently than the Ca2+ exit mode, and inhibition by YM-244769 is [Na+]i-dependent and trypsin-insensitive. These characteristics are similar to those of other benzyloxyphenyl derivative NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The potency of YM-244769 as an NCX1 inhibitor is higher than those of KB-R7943 and SN-6 and is similar to that of SEA0400.

BMP3 expression in the adult rat CNS.

Bone morphogenetic protein-3 (BMP3) is a very unique member of the TGF-ß superfamily, because it functions as an antagonist to both the canonical BMP and activin pathways and plays important roles in multiple biological events. Although BMP3 expression has been described in the early development of the kidney, intestine and bone, little information is available for BMP3 expression in the central nervous system (CNS). We, thus, investigated BMP3 expression in the adult rat CNS using immunohistochemistry. BMP3 was intensely expressed in most neurons and their axons. Furthermore, we found that astrocytes and ependymal cells also express BMP3 protein. These data indicate that BMP3 is widely expressed throughout the adult CNS, and its abundant expression in the adult brain strongly supports the idea that BMP3 plays important roles in the adult brain.

Nicorandil stimulates a Na⁺/Ca²âº exchanger by activating guanylate cyclase in guinea pig cardiac myocytes.

Nicorandil, a hybrid of an ATP-sensitive K(+) (KATP) channel opener and a nitrate generator, is used clinically for the treatment of angina pectoris. This agent has been reported to exert antiarrhythmic actions by abolishing both triggered activity and spontaneous automaticity in an in vitro study. It is well known that delayed afterdepolarizations (DADs) are caused by the Na(+)/Ca(2+) exchange current (I NCX). In this study, we investigated the effect of nicorandil on the cardiac Na(+)/Ca(2+) exchanger (NCX1). We used the whole-cell patch clamp technique and the Fura-2/AM (Ca(2+) indicator) method to investigate the effect of nicorandil on I NCX in isolated guinea pig ventricular myocytes and CCL39 fibroblast cells transfected with dog heart NCX1. Nicorandil enhanced I NCX in a concentration-dependent manner. The EC50 (half-maximum concentration for enhancement of the drug) values were 15.0 and 8.7 µM for the outward and inward components of I NCX, respectively. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a membrane-permeable analog of guanosine 3',5'-cyclic monophosphate (cGMP), enhanced I NCX. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor (10 µM), completely abolished the nicorandil-induced I NCX increase. Nicorandil increased I NCX in CCL39 cells expressing wild-type NCX1 but did not affect mutant NCX1 without a long intracellular loop between transmembrane segments (TMSs) 5 and 6. Nicorandil at 100 µM abolished DADs induced by electrical stimulation with ouabain. Nicorandil enhanced the function of NCX1 via guanylate cyclase and thus may accelerate Ca(2+) exit via NCX1. This may partially contribute to the cardioprotection by nicorandil in addition to shortening action potential duration (APD) by activating KATP channels.

Gamma-delta T cell is essential for allergen-induced late asthmatic response in a murine model of asthma.

BACKGROUND: Gamma-delta (gamma-delta) T cells regulate immune responses at mucosal surfaces. Whether they can modify allergen-induced early (EAR) and late airway responses (LAR) is unknown. OBJECTIVE: We have tested the hypothesis that the gamma-delta T cells enhance allergen-induced airway responses in mice. METHODS: BALB/c wild-type (WT) mice and gamma-delta T cell-deficient (gamma-delta T-cell KO) mice were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 1 and 15, immunized with 1% OVA aerosol on days 29-31, and challenged with 5% OVA or saline on day 33. Enhanced pause (Penh) was measured and BAL fluid was collected after challenge. Serum IgE was measured before challenge. The percentage of interleukin (IL)-4 and interferon (IFN)-gamma producing T cells in splenocytes from sensitized animals was determined by flow cytometry. RESULTS: Both EAR and LAR were observed in OVA-challenged WT mice. LAR but not EAR was inhibited in OVA-challenged gamma-delta T-cell KO mice. Gamma-delta T-cell KO mice showed less eosinophilia in BALF and serum OVA-specific IgE. In the sensitization period, the percentage of IFN-gamma producing alpha-beta T cell in gamma-delta T-cell KO mice was higher than that in WT mice. CONCLUSION: gamma-delta T cells enhance LAR and airway inflammation but not EAR in this model of asthma.

Characterization of SN-6, a novel Na+/Ca2+ exchange inhibitor in guinea pig cardiac ventricular myocytes.

We examined the effect of SN-6, a new benzyloxyphenyl Na(+)/Ca(2+) exchange (NCX) inhibitor on the Na(+)/Ca(2+) exchange current (I(NCX)) and other membrane currents in isolated guinea pig ventricular myocytes using the whole-cell voltage-clamp technique. SN-6 suppressed I(NCX) in a concentration-dependent manner. The IC(50) values of SN-6 were 2.3 microM and 1.9 microM for the outward and inward components of the bi-directional I(NCX), respectively. On the other hand, SN-6 suppressed the outward uni-directional I(NCX) more potently (IC(50) value of 0.6 microM) than the inward uni-directional I(NCX). SN-6 at 10 microM inhibited the uni-directional inward I(NCX) by only 22.4+/-3.1%. SN-6 and KB-R7943 suppressed I(NCX) more potently when intracellular Na(+) concentration was higher. Thus, both drugs inhibit NCX in an intracellular Na(+) concentration-dependent manner. Intracellular application of trypsin via a pipette solution did not change the blocking effect of SN-6 on I(NCX). Therefore, SN-6 is categorized as an intracellular-trypsin-insensitive NCX inhibitor. SN-6 at 10 microM inhibited I(Na), I(Ca), I(K) and I(K1) by about 13%, 34%, 33% and 13%, respectively. SN-6 at 10 microM shortened the action potential duration at 50% repolarization (APD(50)) by about 34%, and that at 90% repolarization (APD(90)) by about 25%. These results indicate that SN-6 inhibits NCX in a similar manner to that of KB-R7943. However, SN-6 at 10 microM affected other membrane currents less potently than KB-R7943.

Electrophysiological effects of SN-6, a novel Na+/Ca2+ exchange inhibitor on membrane currents in guinea pig ventricular myocytes.

We examined the effect of SN-6 on the Na+/Ca2+ exchanger (NCX) current (I(NCX)) and other membrane currents in isolated guinea pig ventricular myocytes using the whole-cell voltage clamp technique. SN-6 suppressed the bidirectional I(NCX) in a concentration-dependent manner. The IC50 values of SN-6 were 2.3 microM and 1.9 microM for the outward and inward components of the bidirectional I(NCX), respectively. On the other hand, SN-6 suppressed the unidirectional outward I(NCX) more potently than the inward I(NCX), with an IC(50) value of 0.6 microM. SN-6 at 10 microM inhibited the unidirectional inward I(NCX) by only 22.4 +/- 3.1%. SN-6 suppressed I(NCX) more potentially when intracellular Na+ concentration became higher. SN-6 inhibited I(Na), I(Ca), I(Kr), I(Ks), and I(K1) by about 13%, 34%, 33%, 18%, and 13%, respectively. SN-6 shortened the action potential duration (APD) by about 34% and 25% at APD(50) and APD(90), respectively. These results indicate that SN-6 inhibits NCX in a similar manner to that of KB-R7943. SN-6 and KB-R7943 inhibit the unidirectional outward I(NCX) more potently than the unidirectional inward I(NCX). Both drugs inhibit NCX in an intracellular Na+ concentration-dependent manner. However, SN-6 affected other membrane currents less potently than KB-R7943.

A novel secretory factor, Neurogenesin-1, provides neurogenic environmental cues for neural stem cells in the adult hippocampus.

Neurogenesis occurs in restricted regions in the adult mammalian brain, among which the neurogenesis in the hippocampal dentate gyrus plays the crucial role in learning and memory. To date, little is known about neurogenic cues, which result in the neuronal fate adoption of neural stem cells residing in neurogenic regions, especially neurogenic cues in adult hippocampal neurogenesis. In the present study, we show that hippocampal astrocytes and also dentate granule cells adjacent to neural stem cells secrete a newly cloned novel secretory factor, Neurogenesin-1. This protein contains three cysteine-rich domains and a unique sequence and contributes to neuronal differentiation of neural stem cells in the adult brain by preventing the adoption of a glial fate. Furthermore, the neurogenic activity detected in the hippocampal culture medium was markedly suppressed by the administration of an anti-Neurogenesin-1 antibody. These findings suggest endogenous mechanisms that induce adult hippocampal neurogenesis and propose an innovative treatment for the neurodegenerative diseases that cause loss of hippocampal neurons.